WO2018149282A1

WO2018149282A1 - Method for heterologous expression of epothilone

Info

Publication number: WO2018149282A1
Application number: PCT/CN2018/074229
Authority: WO
Inventors: 丁晓明; 禹育聪; 唐标; 张霖; 王慧敏; 卞小莹; 李越中; 张友明; 赵国屏
Original assignee: 复旦大学
Priority date: 2017-02-20
Filing date: 2018-01-26
Publication date: 2018-08-23
Also published as: CN108456703A; CN108456703B

Abstract

Disclosed is a method for heterologous expression of an epothilone, comprising introducing an epothilone gene cluster into host bacteria, while supplementing an epothilone precursor synthesis pathway combining with the introduction of the related tRNA gene and insertion of a promoter. The method can substantially increase the expression quantity of the epothilone, and the yield can be improved by about four orders of magnitude and can reach 8.5 mg/L. In addition, also provided are a genetic engineering strain for heterologous expression of the epothilone and a method for producing the epothilone.

Description

Method for heterologous expression of epothilone

Technical field

The invention relates to the field of biological expression, in particular to a method for heterologous expression of epothilone and related genetic engineering strains.

Background technique

Epothilines, produced by Fermentation of Sorangium cellulosum , is a novel antitumor drug similar to paclitaxel with microtubule stabilization. There are now five epothilones and derivatives used in the clinic, in which Patupilone (Epothilone B) and KOS-862 (Epothilone D) are fermentation products, Ixabepilone and BMS-310705 are Eb. A chemical modification of B, ZK-EPO is a chemically synthesized drug. Ixabepilone was approved by the US FDA in October 2007 for advanced breast cancer treatment. At present, the main fermentation strain of epothilone is the original strain of cellulose bacillus, and the natural strain mainly produces epothilone A and B.

Polyglycans [ Polyangium ] brachysporum DSM 7029 (=K481-B101 = ATCC 53080) was isolated from soil in Greece in 1988. DSM 7029 strain can produce antifungal and tumor drug glibenclamide as proteasome inhibition. Agent, which is a natural product of a heterozygous NRPS/PKS (non-ribosomal polypeptide synthase/polyketone synthase) type. DSM 7029 strain taxonomic status has not been determined, it is burkholderiales (Burkholderiales) by 16S rDNA analysis. The strain grew faster than the original production strain of Epothilone, the cellulite, and the model Myxobacteria, Myxococcus xanthus . Single colonies were observed in two days. Studies have found that this strain can heterologously express epothilone.

Through the prior art, the original production strain of cellulose bacterium can be optimized by mutagenesis to reach about 100 mg / L, but the fermentation cycle exceeds 20 It is easy to pollute and it is difficult to improve the space. Eptomycin could not be efficiently expressed by other model bacteria such as E. coli or DSM 7029 before transformation, and the yield could only reach about 1 ug/L. . Therefore, there is a need to develop a method for efficiently heterologous expression of epothilone and a genetically engineered strain for production.

Summary of the invention

In order to overcome the problem of low expression efficiency of epothilone in the prior art, one aspect of the present invention provides a A method of heterologous expression of epothilone. In a specific embodiment, the method introduces an epothilone gene cluster into a host strain while supplementing the epothilone precursor synthesis pathway. Among them, the epothilone gene cluster is derived from Cellulose bacillus.

Further, the host bacteria belong to the genus Burkholderiales .

Further, the host strain is a strain of Burkholderia DSM 7029.

Further, the precursor synthesis pathway is a synthetic route of S-methylmalonyl-CoA.

Further, the synthetic route for supplementing the S-methylmalonyl-CoA is to supplement the PCC pathway, the MatB pathway, and the mutase. - one or more of the isomerase pathways.

Preferably, the synthetic route to supplement the S-methylmalonyl-CoA is to supplement the PCC pathway, the MatB pathway, and the mutase. - Isomerase pathway.

Further, the PCC pathway is by adding propionyl-CoA carboxylase (propionyl-CoA carboxylase) ) to complement; MatB pathway by adding malonyl-CoA/methylmalonyl-CoA synthetase (malonyl-CoA/methylmalonyl-CoA) Synthetase) to complement; mutase-isomerase pathway by adding methylmalonyl-CoA isomerase (methylmalonyl-CoA epimerase ) to add.

Further, propionyl-CoA carboxylase is the accA1 / pccB or pccA / pccB gene of S. coelicolor A3(2).

Further, the methylmalonyl-CoA isomerase is an epi gene of S. coelicolor A3(2).

Further, the malonyl-CoA/methylmalonyl-CoA synthetase is the matB gene of S. coelicolor A3(2).

Further, a tRNA gene has also been introduced into the host strain.

Further, the tRNA genes are Arg anti-GCG, Arg anti-TCG, Gln One or more of the anti-CTG and Glu anti-CTC genes.

Further, Arg anti-GCG, Arg anti-TCG, Gln anti-CTG and The Glu anti-CTC gene is derived from Myxococcus xanthus DK 1622.

Further, a promoter sequence is added in front of one or more genes in the epothilone gene cluster.

Preferably, a promoter sequence is added before one or more of the 6 genes epoA , epoB , epoC , epoD , epoE and epoF in the epothilone gene cluster.

Further preferably, a promoter sequence is added before each of the 6 genes epoA , epoB , epoC , epoD , epoE and epoF in the epothilone gene cluster.

Further, the above promoter is PKan.

Further, a promoter sequence is added by resplicing of the gene.

Further, splicing was performed using the Bxb1 integrase splicing technique.

Another aspect of the invention provides a genetically engineered strain that heterologously expresses epothilone. In a specific embodiment, the epothilone gene cluster is introduced into the genetically engineered strain, supplemented with an epothilone precursor synthesis pathway. Among them, the epothilone gene cluster is derived from Cellulose bacillus.

Further, the basic strain of the genetically engineered strain is a Burkholderiales DSM 7029 strain.

Further, the basic strain is supplemented with a synthetic route of the epothilone precursor S-methylmalonyl-CoA.

Further, the synthetic pathway of S-methylmalonyl-CoA includes the PCC pathway, the MatB pathway, and the mutase- Isomerase pathway.

Further, on the basis of strains, by adding Streptomyces coelicolor (S. coelicolor) A3 (2) of accA1 / pccB added to the PCC pathway; by addition of Streptomyces coelicolor (S. coelicolor) A3 (2) of the epi The gene complements the mutase-isomerase pathway; the matB pathway is complemented by the addition of the matB gene of S. coelicolor A3(2).

Further, a tRNA gene is also added to the base strain.

Further, the tRNA genes are Arg anti-GCG, Arg anti-TCG, Gln anti-CTG and Glu anti-CTC genes.

Further, a promoter sequence is added to one or more genes of the epothilone gene cluster.

Preferably, a promoter sequence is added before each of the 6 genes epoA , epoB , epoC , epoD , epoE and epoF in the epothilone gene cluster.

Further, the promoter sequences are sequentially spliced in the order of epoA , epoB , epoC , epoD , epoE to epoF by resplicing .

Further, the promoter is PKan.

Further, in a preferred embodiment of the present invention, the genetically engineered strain heterologously expressing epothilone is Polyangium brachysporum MMR11, accession number CCTCC M 2017037, on January 19, 2017 Preserved in the China Center for Culture Collection Management.

Yet another aspect of the present invention provides a A method of producing epothilone. In a specific embodiment, it provides a strain as described above which is fermented in a fermentation medium at 30 ± 2 °C.

Further, the fermentation medium is CYMG fermentation medium, and the formula per liter is 8 g of casein, 4 g of yeast extract, magnesium chloride hexahydrate. 4.06 g , 50% glycerol 10 ml , trace element 1 ml, sodium acetate 50 mg, sodium propionate 100 mg, methylmalonic acid 100 mg, cysteine 2.5 Mg, serine 5 mg, XAD-16 macroporous adsorption resin wet weight 1%, adjusted pH 7.0-7.5.

Further, the fermentation time was 3 days.

The method for heterologous expression of epothilone in the present invention, by analyzing the host strain genome and the epothilone gene cluster, the epothilone precursor S-methylmalonyl-CoA lacking in the host strain The synthetic pathway and expression elements such as tRNA and promoter suitable for the epothilone gene cluster have been modified to increase the yield of epothilone. In a preferred embodiment, the epothilone is in the modified DSM 7029 The strain is highly expressed, and the yield is increased by about 4 orders of magnitude to 8.5 mg/L, which is 105 times that of the strain containing only the unmodified gene cluster, which is in line with the industrial production of epothilone.

DRAWINGS

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a supplementary schematic diagram of the synthesis pathway of the precursor S-methylmalonyl-CoA in a specific embodiment of the present invention.

2 is a specific embodiment of the present invention, a precursor S-methylmalonyl-CoA synthesis pathway and tRNA After the addition of the epothilone production plot.

Figure 3 is a transcriptome peak map of the Ebomycin gene cluster of the MMR1 strain containing the Epothilone gene cluster in a specific embodiment of the present invention ( T is the chain of justice and F is the antisense strand).

Figure 4 is a schematic diagram showing the resplicing and promoter addition of the epothilone synthetic gene cluster in one embodiment of the present invention.

Figure 5 is a map of plasmid pST-BSD-epo in a specific embodiment of the present invention.

Figure 6 is a graph showing the yield of epothilone after resplicing the epothilone biosynthetic gene cluster in a specific embodiment of the present invention.

detailed description

The invention is further illustrated by the following examples, which are intended to be illustrative only and not to limit the scope of the invention.

The invention concludes the reason that the low yield of heterologous expression of epothilone in the conventional host strain is low by genomic analysis of the host strain and the analysis of the epothilone gene cluster, and the host strain is modified according to these factors, and the E. eutropha has been improved. A method for heterologous expression and provides related engineered strains.

1. Characterization of DSM 7029 strain and analysis of epothilone gene cluster characteristics

The genome of DSM 7029 was not previously sequenced. After sequencing and analysis of the genome of DSM 7029, DSM was discovered. The genomic GC content of 7029 was 67.51%, which is close to the GC content of the 56 kb gene cluster of epothilone.

The DSM 7029 genome contains multiple NRPSs and PKSs, of which the largest CDS encodes a non-ribosomal peptide synthetase ( AAW51_3371 ) with a size of 32,469 bp. The Epothilone gene cluster derived from Sorangium cellulosum encodes a hybrid NRPS/PKS with a size of 56 kb, including 9 PKS modules, 1 NRPS module, and 1 P450 oxidase. The complete epothilone gene cluster includes epoA (sequence as shown in SEQ ID No. 1), epoB (sequence as shown in SEQ ID No. 2), epoC (sequence as shown in SEQ ID No. 3), epoD ( The sequence is as shown in SEQ ID No. 4, epoE (sequence as shown in SEQ ID No. 5), epoF (sequence as shown in SEQ ID No. 6), and epoK 7 genes. Among them, P450 oxidase (EpoK) can catalyze the conversion of epothilone C\D to epoxy structure to epothilone A\B. In the case of only 6 genes of epoA , epoB , epoC , epoD , epoE and epoF , epothilones C and D are produced. The epothilone gene cluster derived from Sorangium cellulosum lacks the necessary promoter sequence for DSM 7029 expression.

In the production of epothilone, the epothilone gene cluster derived from Sorangium cellulosum may be selected, or the epothilone gene cluster derived from other strains may be selected.

tRNA analysis:

For tRNA statistics of DSM 7029 strain and codon preference analysis of epothilone (using tRNAscan-SE V1.23 analysis), DSM 7029 strain lacks the main expression of epothilone 4

tRNAs: Arg anti-GCG, Arg anti-TCG, Gln anti-CTG and Glu anti-CTC. DSM 7029 strain and Myxococcus xanthus , Sorangium cellulosum

The tRNAs were compared (as shown in Table 1) and the same was found for three of the above tRNAs. Supplement tRNAs It is important for the production of epothilone.

Table 1. Comparison of the types and quantities of tRNA in the genomes of M. xanthus DK1622 , S. cellulosum So0157-2 and DSM 7029 strains

	*Myxococcus xanthus* DK1622	Sorangium cellulosum So0157-2	DSM 7029
Amino acid	Anti-codon	Quantity	Anti-codon	Quantity	Anti-codon	Quantity
Ala	CGC	1	CGC	2	CGC	1
Ala	GGC	2	GGC	1	GGC	1
Arg	ACG	1	ACG	2	ACG	2
Arg	CCG	1	CCG	1	CCG	2
Arg	CCT	1	CCT	1	CCT	1
Arg	GCG	1	GCT	1	-	-
Cys	GCA	1	GCA	1	GCA	1
Gln	CTG	1	CTG	1	-	-
Glu	CTC	1	CTC	1	-	-
Gly	CCC	1	CCC	1	CCC	1
Gly	GCC	3	GCC	3	GCC	3
Ile	GAT	5	GAT	2	GAT	4
Leu	CAA	2	CAA	1	CAA	1
Leu	CAG	2	CAG	1	CAG	1
Leu	GAG	1	GAG	1	GAG	1
Lys	CTT	1	CTT	2	CTT	1
Met	CAT	6	CAT	4	CAT	3
Phe	GAA	1	GAA	1	GAA	3
Pro	CGG	1	CGG	1	CGG	1
Pro	GGG	1	GGG	2	GGG	1
Ser	CGA	1	CGA	2	CGA	1
Ser	GCT	1	-	-	GCT	2
Ser	GGA	1	GGA	1	GGA	1
Thr	CGT	1	CGT	1	CGT	1
Trp	CCA	1	CCA	1	CCA	2
Val	CAC	1	CAC	1	CAC	1
Val	GAC	1	GAC	1	GAC	1
Xxx	Xxx	1	-	-	Xxx	1

- : Missing

Analysis of precursor synthesis pathways:

The epothilone gene cluster is a PKS/NRPS type gene cluster, and the synthetic precursor of epothilone A includes 1 molecule of acetate, 4 molecules. S-methylmalonyl-CoA, 4 molecules of malonyl-CoA, a molecule of SAM-derived methyl carbon and a molecule of cysteine. One molecule of malonyl-CoA when epothilone B is synthesized Replaced by methylmalonyl-CoA. Among them, S-methylmalonyl-CoA is an important synthetic precursor of epothilone.

S-methylmalonyl-CoA has the following synthetic routes:

1) PCC pathway: propionate is catalyzed by propionyl-CoA synthetase ( prpE , EC: 6.2.1.17) to synthesize propionyl-CoA, followed by propionyl-CoA carboxylase (propionyl-CoA carboxylase , pccA / pccB , EC: 6.4.1.3) Synthesis of S-methylmalonyl-CoA ((2S)-methylmalonyl-CoA);

2) MatB pathway: substrate methylmalonic acid by malonyl-CoA/methylmalonyl-CoA synthetase (malonyl-CoA/methylmalonyl-CoA synthetase, matB , EC: 6.2.1.-) Methylmalonyl-CoA;

3) The mutase-epimerase pathway: succinyl-CoA undergoes methylmalonyl-CoA mutase ( mcmA , EC: 5.4.99.2) generating R- methylmalonyl-CoA A ((2 R) -methylmalonyl- CoA), and then produced from the S- methyl methylmalonyl-CoA isomerase A (methylmalonyl-CoA epimerase, epi, 5.1.99.1) Malonyl-CoA (( 2S )-methylmalonyl-CoA).

Annotated alignment of the genome of DSM 7029 strain revealed that DSM 7029 strain lacks intact S-methylmalonyl-Coenzyme A synthetic pathway that lacks the PCC pathway and the mutase-isomerase pathway.

Based on the above analysis, consider adding a suitable tRNA from the path of supplementation of the epothilone precursor. Modifications were made with the addition of a promoter to increase the yield of epothilone.

The invention will be further explained below in conjunction with specific embodiments.

The method of operation in the examples, unless otherwise specified, can be accomplished by conventional techniques in the art.

Restriction enzymes were purchased from Thermo Fisher Scientific; Ex Taq, GC buffer I / II, PrimerSTAR TM HS DNA polymerase, λ-EcoT14 Marker available from TakaRa-

Bioengineering (Dalian) Co., Ltd.; DNA The agarose gel recovery kit and the bacterial genome extraction kit were purchased from Shanghai Jierui Biological; the PCR product recovery kit was purchased from the bioengineering bioengineering;

CloneExpress MultiS, CloneExpress II, Phanta Max Super-Fidelity DNA Polymerase was purchased from Nanjing Nuoweizan; XAD-16 (macroporous resin) was purchased from Shanghai Mospeed Scientific Equipment; Epothilone B was purchased from Toronto Research Chemicals; Epothilone A, Epothilone C, Epothilone D was purchased from Dalian Meilun Bio; chromatography grade methanol, chromatographic grade acetonitrile was purchased from Germany MERCK ; Ultimate XB-C18, 5μm, 4.6×250mm purchased from Yuexu Technology (Shanghai) Co., Ltd.; casein 胨 purchased from the United States BD Company; yeast extract was purchased from OXIOD; magnesium chloride and glycerol were purchased from bioengineering.

antibiotic:

name	Solvent	Stock solution	Working concentration
Ampicillin (Ampicillin)	ddH ₂ O	100 mg/mL	100 μg/mL
Kanamycin	ddH ₂ O	50 mg/mL	50 μg/mL
Apramycin	ddH ₂ O	50 mg/mL	50 μg/mL
Spectinomycin	ddH ₂ O	50 mg/mL	50 μg/mL

Example 1 Adding a precursor metabolic pathway pathway gene and tRNA to increase the production of epothilone

As previously analyzed, the DSM 7029 strain lacks a complete S-methylmalonyl-CoA synthesis pathway with no PCC Pathway and mutase - isomerase pathway. Therefore, consider increasing the production of epothilone by supplementing the pathway of S-methylmalonyl-CoA synthesis. Shown. The method of operation is to first construct a plasmid containing the epo gene cluster, and transfer it to the DSM 7029 strain, and then construct a plasmid containing different complement pathway genes and /tRNA genes, and then transfer to the above-mentioned containing Among the DSM 7029 strains of the epo gene cluster, engineered strains were obtained.

The construction of plasmid 1 (containing the epo gene cluster) was as follows: plasmids Cosmid 10 and Fosmid 3B11 were screened for the Soybean cellulase genome So0157-2 library (see Allopatric integrations selective change host transcriptomes, leading to varied expression efficiencies of exotic genes in Myxococcus xanthus, Microb Cell Fact [J] , 2015; 14:. 38.5 kb fragment 105. the front portion), respectively, with epothilone biosynthetic gene cluster from epoA to epoD and the rear portion of epoC to epoF The 34.4 kb fragment of the downstream fragment, which has a coverage of 6.5 kb. Whole gene synthesis PCR targeting fragment 1 (SEQ ID No. 7) and fragment 2 (SEQ ID No. 8) (wherein the p15A replicon, the resistance gene and the att site are partially located on the fully synthesized fragment 1 and fragment 2) Plasmid pZLE21 (containing attB0 and attP6 sites) and pZLE22 (containing attB6 and attP15 sites) were obtained by PCR targeting with plasmids Cosmid 10 and Fosmid3B11, respectively. Finally, pZLE21, pZLE22 and pZLE19 (including attB15 and attP0 sites) utilize the phi BT1 integrase reaction (plasmid pZLE19 and integrase reaction reference Tandem assembly of the epothilone biosynthetic gene cluster by in vitro site-specific recombination, Sci Rep. 2011; 1:141. doi: 10.1038/srep00141), the plasmid pZL-epo was obtained, which contained all the epothilone biosynthetic gene clusters. Wherein, the attB0 site is represented by SEQ ID No. 9, the attP6 site is represented by SEQ ID No. 12, the attB6 site is represented by SEQ ID No. 11, and the attP15 site is represented by SEQ ID No. 18.

Transfer plasmid pZL-epo containing the Epothilone gene cluster into GBred strain (purchased from Gene Bridges) ), preparation of electroporation competent state, using primer BSD-epo-F/R (as shown in SEQ ID No. 71 and 72) to plasmid pBSD containing transposase (Reference) Nucleic Acids Res. 2008 Oct;36(17):e113. doi: 10.1093/nar/gkn499 , the plasmid can be inserted by transposition The DSM7029 genome is used as a template, and the amplified product is electroporated into GBred/pZL-epo. The plasmid containing the transposase and the complete epothilone gene cluster can be obtained by homologous recombination. pBSD-epo , named plasmid 1 .

AnacA1 / pccB or pccA / pccB gene derived from S. coelicolor A3(2) (purchased from ATCC, number ATCC BAA-471) was added according to the supply pathway of S-methylmalonyl-CoA. Supplemented the PCC pathway; added the epi gene derived from S. coelicolor A3(2) to complement the mutase-isomerase pathway; added Streptomyces coelicolor ( S. The matB gene of coelicolor A3(2) ) adds a copy that can express another malonyl-CoA/methylmalonyl-CoA synthetase.

Specifically, the accA1-F/R (as shown in SEQ ID No. 21 and 22) was used as a primer to amplify the accA1 gene (shown as SEQ ID No. 31) using the S. cerevisiae genome as a template , pccA-F /R (shown as SEQ ID No. 23 and 24) amplified the pccA gene (shown as SEQ ID No. 32) for the primer, as pccB-F/R (as shown in SEQ ID No. 25 and 26) The pccB gene was amplified for the primer (as shown in SEQ ID No. 33); the epi gene was amplified with primer-F/R (as shown in SEQ ID No. 27 and 28) as primer (eg SEQ ID No. 34). The matB gene (shown as SEQ ID No. 35) was amplified using MatB-F/R (shown as SEQ ID No. 29 and 30) as a primer.

Using plasmid pBSD as a template, BSD-F/R (eg SEQ ID No. 19 and 20) Shown as a primer to amplify a vector fragment, ligated to the above gene using a seamless ligation technique.

As previously analyzed, the DSM 7029 strain lacks 4 tRNAs that predominantly express epothilone: Arg anti-GCG, Arg anti-TCG, Gln anti-CTG and Glu anti-CTC. Therefore, consider adding these 4 tRNAs To increase the yield of epothilone.

Four genes derived from S. aureus were synthesized by whole gene, and the Arg anti-GCG sequence was SEQ ID As shown in No. 36, the Arg anti-TCG sequence is as shown in SEQ ID No. 37, and the Gln anti-CTG sequence is as SEQ ID No. 38. The Glu anti-CTC sequence is shown in SEQ ID No. 39, and the whole whole gene synthesis tRNA sequence of the four genes is SEQ ID No. 70. Shown and cloned with tRNA-F/R (as shown in SEQ ID No. 40 and 41) as primers.

Synthesizing tRNA in front of each of the genes in the amplified five of the above precursor metabolic pathways Whole gene (in the whole gene synthesis, according to Arg anti-GCG, Arg anti-TCG, Gln anti-CTG and Glu anti-CTC The sequence is synthesized as a whole fragment) with the promoter PKan (sequence shown in SEQ ID No. 42), and then constructed into a vector containing the transposase pBSD by seamless ligation. On. Among them, 7 plasmids were constructed: plasmid 2 (pccA+pccB), plasmid 3 (pccA+pccB+tRNA), plasmid 4 (acc1+pccB) ), plasmid 5 ( accA1 + pccB + tRNA ), plasmid 6 ( accA1 + pccB + epi + tRNA ), plasmid 7 ( accA1+pccB+matB+tRNA ), plasmid 8 ( accA1+pccB+epi+matB+tRNA ) to see the effect of different combinations of additions on epothilone production. First, the constructed plasmid 1 was transformed into DSM7029 to obtain the recombinant MMR1 containing the epothilone gene cluster, and the above plasmid 2-8 was taken. MMR1 was transformed to obtain each of the recombinant strains Mb2-8 expressing Epothilone. The specific transformation process is as follows: 3 microliters of the plasmid to be transformed is added to DSM 7029 or transferred to the epothilone gene cluster. MMR1 competent cells, mixed and added to a 2mm electric shock cup, 2500V for electrotransformation, and after 3 hours of activation, coated with Kanamycin or apramycin ( Apramycin) plate. Positive clones were picked on the plates after 2 days.

The fermentation of epothilone is carried out using 500 ml of CYMG fermentation medium in the form of Casitone (BD). Company ) 8 g , yeast extract (OXOID company ) 4 g , magnesium chloride hexahydrate 4.06 g , 50 % glycerol 10 ml , trace element 1 ml , sodium acetate 50 Mg/L, sodium propionate 100 mg/L, methylmalonic acid 100 mg/L, cysteine 2.5 mg/L, serine 5 mg/L, XAD-16 macroporous adsorption resin wet weight 1%, add water to 1L, adjust pH 7.0-7.5, and heat sterilization at 121 °C for 20 min. Fermentation temperature is 30 °C, shaker speed is 200 rpm, fermentation 3 Day.

The above trace elements are: 100 mL of water, dissolved manganese chloride tetrahydrate 0.79 g, zinc sulfate heptahydrate 0.15 g , copper sulfate pentahydrate 0.64 g, ferrous sulfate heptahydrate 0.11 g, used as a mother liquor.

After the fermentation is finished, the resin is poured into a 100-mesh standard sieve and washed several times. After drying, 25 ml of methanol is added, 30 Analyze twice at °C for 12 hours each time. Concentrate twice with methanol solution, mix and filter by EHPLC-MS/MS for Ebomycin A, B, C and D The yield is quantified.

As shown in Figure 2, the original strain DSM 7029 did not have epothilone production. The recombinant strain MMR1 was obtained by adding the epothilone gene cluster (epo gene cluster) to DSM 7029, and the production of epothilone C and D reached 61.27 μg/L and 18.76 μg/L. On the basis of this, the pccA/pccB/tRNAs pathway was added to obtain the strain MMR3, and the total yield was increased by 10%. On the basis of supplementing the accA1/pccB and accA1/pccB/tRNAs pathways, the strains MMR4 and MMR5 were obtained, and the total yield was doubled. At 100% and 130%, the production of epothilone C and D reached 129.54 μg/L and 59.35 μg/L. Supplemented accA1 / pccB / tRNAs complement pathway on the basis of each epi genes and gene, the strain MMR6 MatB and MMR7, epothilone production continued to rise significantly, epothilone A, B, C and D production reached 65.57 μg / L, 58.35 μg/L, 508.30 μg/L, 466.40 μg/L and 19.35 μg/L, 3.39 μg/L, 225.20 μg/L, 47.33 μg/L. When the accA1-pccB- tRNAs- epi-matB pathway was supplemented to obtain the strain MMR8, the yield reached the highest, and the production of epothilone A, B, C and D reached 45.85 μg/L, 62.54 μg/L, 399.12 μg/L. With 1101.03 μg/L, the total yield reached 1.6 mg/L, which is 20 times that of the strain containing only the epo gene cluster.

Example 2 The epo gene cluster was re-spliced to add a promoter to increase the yield of epothilone

After transcriptome sequencing of the MMR1 strain containing the Epothilone gene cluster, it was found that the expression levels of each gene in the Epothilone gene cluster were very low (Fig. 3 ), low levels of gene expression may be another key factor limiting the production of epothilone.

In view of the above, in order to improve the expression efficiency of the epothilone synthetic gene cluster in DSM7029, the Eptomycin gene clusters epoA , epoB , epoC , epoD , epoE and epoF were performed using Bxb1 integrase splicing technology. Figure 4 shows the re-splicing. In front of each gene in the epothilone gene cluster, a promoter is added to increase the expression level of each gene in the gene cluster. Try to increase the yield of epothilone by adding a promoter.

Specifically, the plasmid pSB1A3 (derived from iGEM, http://parts.igem.org/Part:pSB1A3) was used as a template, and epo1A3-F/R (as shown in SEQ ID No. 43 and 44) was used as a primer clone Amp. Sequence, using plasmid pSB3K5 (derived from iGEM at http://parts.igem.org/Part:pSB3K5) as a template, epo3K5-F/R (shown as SEQ ID No. 45 and 46) as primer clone P15A sequence The two fragments were ligated seamlessly to obtain the plasmid p-vector (containing four restriction endonuclease sites for EcoR I , Xba I , Spe I and Pci I). Using the plasmid pZL-epo containing the epothilone gene cluster as a template, epoA-F/R (as shown in SEQ ID No. 47 and 48) is the primer clone epoA gene, epoB-F/R (eg SEQ ID No.). The primers cloned the epoB gene, and epoC-F/R (as shown in SEQ ID No. 51 and 52) is the primer clone epoC gene, epoF-F/R (as shown in SEQ ID Nos. 57 and 58). The primers were cloned into the epoF gene, using epo-vector-F/R (as shown in SEQ ID No. 73 and 74) as a primer, p-vector as a template to amplify the vector fragment, and the above genes were seamlessly connected. The clones were cloned to obtain pEpoA, pEpoB, pEpoC and pEpoF.

The plasmid pZL-epo containing the epothilone gene cluster was transferred into the GBred strain to prepare an electrotransport competent state, using the intermediate vector p-vector as a template, epoD-F/R (as shown in SEQ ID No. 53 and 54) and epoE-F/R (shown in SEQ ID No. 55 and 56) is a primer that amplifies an intermediate vector linearized fragment containing a homology arm and then electroporates into a GBred/pZL-epo strain by homologous recombination, ie Plasmids pEpoD and pEpoE containing epoD and epoE were obtained.

The plasmids pEpoA, pEpoB, pEpoC, pEpoD, pEpoE and pEpoF containing the epothilone gene were digested with restriction enzymes EcoR I and Xba I. Using pBSD as a template, the primers pKan-F/R (as shown in SEQ ID No. 59 and 60) are primer amplification promoter sequences, and the PKan promoter is digested with restriction endonucleases EcoR I and Spe I. The enzyme fragments were recovered. The double-digested epothilone gene and the PKan promoter fragment were ligated to obtain the epothilone gene plasmid pPKan-EpoA, pPKan-EpoB, pPKan-EpoC, pPKan-EpoD, pPKan-EpoE with promoter added. And pPKan-EpoF.

The plasmid pPKan-EpoB was digested with restriction endonucleases EcoR I and Xba I, and the plasmid pPKan-EpoA was digested with restriction endonucleases EcoR I and Spe I to obtain pPKan-EpoA-PKan-EpoB. The plasmid pPKan-EpoF was digested with restriction endonucleases EcoR I and Xba I, and the plasmid pPKan-EpoE was digested with restriction endonucleases EcoR I and Spe I to obtain pPKan-EpoE-PKan-EpoF. The plasmid pPKan-EpoC was digested with the restriction enzymes EcoR I and Xba I, and the plasmid pPKan-EpoA-PKan-EpoB was digested with the restriction enzymes EcoR I and Spe I to obtain pPKan-EpoA-PKan-EpoB. -pPKan-EpoC. The restriction endonucleases EcoR I and Xba I were used to digest the plasmids pPKan-EpoA-PKan-EpoB-pPKan-EpoC, pPKan-EpoD and pPKan-EpoE-PKan-EpoF, respectively, and ligated to attB0 (as shown in SEQ ID No. 9). , attB13 (shown as SEQ ID No. 15) and attB7 (shown as SEQ ID No. 13), resulting in plasmids pattB0-PKan-EpoA-PKan-EpoB-pPKan-EpoC, pattB13-PKan-EpoD and pattB7-PKan-EpoE-PKan-EpoF. The restriction endonucleases Spe I and Pci I were used to digest the plasmids pattB0-PKan-EpoA-PKan-EpoB-pPKan-EpoC, pattB13-PKan-EpoD and pattB7-PKan-EpoE-PKan-EpoF, respectively, and ligated to attP13 (eg SEQ ID No. 16), attP7 (shown as SEQ ID No. 14) and attP15 (shown as SEQ ID No. 18), yielding pattB0-PKan-EpoA-PKan-EpoB-pPKan-EpoC-attP13 , pattB13-PKan-EpoD-attP7 and pattB7-PKan-EpoE-PKan-EpoF-attP15.

Whole gene synthesis attP0 - ccdB - attB15 fragment (as shown in SEQ ID No. 61), using the synthetic sequence as a template, ccdB-F/R (as shown in SEQ ID No. 62 and 63) as a primer amplification fragment; pBSD is the template, and the primers ccdB-vector-F/R (shown as SEQ ID No. 64 and 65) amplify the fragments, and the two are ligated using a seamless clone to obtain the plasmid pBSD-ccdB. Using pBSD-ccdB as a template, the primer BSD-ccdB-F/R (as shown in SEQ ID No. 66 and 67) is a primer-amplified fragment; using plasmid pST-ccdB as a template, primer ST-F/R (such as SEQ Amplification fragments were shown in ID No. 68 and 69, and the two were seamlessly ligated to obtain plasmid pST-BSD.

Vector plasmid pST-BSD 0.5 μL, pattB0-PKan-EpoA-PKan-EpoB- pPKan-EpoC-attP13 plasmid 2 μl, pattB13-PKan-EpoD-attP7 plasmid 2 μl, pattB7-PKan-EpoE-PKan- EpoF-attP15 plasmid 1.5 μl, Bxb 1 integrase 1 μl, reaction at 30 °C for 20 hours, the reaction system was transformed with high temperature and proteinase K, and the correct clone was screened on the antibiotic kanamycin plate. The correct plasmid pST-BSD-epo map is shown in Figure 5.

Five microliters of the pST-BSD-epo plasmid was electroporated into the DSM 7029 strain to obtain a recombinant strain MMR10 containing the engineered epothilone gene cluster to which the promoter was added. The plasmid containing accA1-pccB- tRNAs -epi -matB was electroporated on the basis of the strains of the engineered gene cluster, and the strain MMR11 with high yield of epothilone was obtained.

Use strains MMR1, MMR10, MMR8, MMR11 with 500 mL CYMG Fermentation medium, fermentation temperature was 30 °C, shaker speed was 200 rpm, and fermentation was carried out for 3 days.

After the end of the fermentation, the resin is concentrated in a standard sieve of 100 mesh for several times, and after drying, 25 ml of methanol is added, 30 Analyze twice at °C for 12 hours each time. The methanol solution was concentrated twice and mixed, and the yield of epothilone C and D was quantified by UHPLC-MS/MS after filtration.

As shown in Figure 6, the DSM 7029 fermentation broth was not produced by epothilone. After the unspliced Epothilone gene cluster was added to DSM 7029, the yield of Epothilone C and D of strain MMR1 reached 61.27 μg/L and 18.76 μg/L. The production of epothilone was detected when the recombined add-on promoter of the epothilone gene cluster was transferred into DSM 7029. The yields of epothilone C and D were 83.63 μg/L and 25.38 μg/L. The total amount has increased by 36%. When tRNAs and accA1-pccB-epi-matB genes were added to strains containing unmodified Epothilone gene clusters, the yields of epothilone C and D reached 399.12 μg/L and 1101.03 μg/L. When tRNAs and the above-mentioned precursor-related genes were added to the strain containing the respliced and added promoter, the yield was greatly increased, and the yields of epothilone C and D were 4,721.47 μg/L and 3,812.25 μg/L, and the total yield was only The strain containing the unmodified gene cluster increased by 105 times.

In summary, we have combined the expression of the epothilone gene cluster by adding the PCC pathway, the MatB pathway, the mutase-isomerase pathway, etc., which synthesize S-methylmalonyl-CoA in Burkholderiales DSM 7029. The tRNAs and promoters were modified to enable efficient expression of epothilone in DSM 7029. The shake flask yielded 8.5 mg/L in 3 days, which was basically in line with the industrial production of epothilone.

Claims

A method for heterologous expression of epothilone, characterized in that an epothilone gene cluster is introduced into a host strain, and an epothilone precursor synthesis pathway is supplemented.
The method of heterologously expressing epothilone according to claim 1, wherein the host bacterium belongs to Burkholderiales.
The method of heterologously expressing epothilone according to claim 1, wherein the host strain is a Bordetella DSM 7029 strain.
The method of heterologously expressing epothilone according to claim 3, wherein the precursor synthesis pathway is a synthetic pathway of S-methylmalonyl-CoA.
The method for heterologously expressing epothilone according to claim 4, wherein the synthetic route for supplementing the S-methylmalonyl-CoA is supplemented with a PCC pathway, a MatB pathway, and a mutase-differentiation One or more of the enzyme pathways.
The method for heterologously expressing epothilone according to claim 4, wherein the synthetic route for supplementing the S-methylmalonyl-CoA is supplemented with a PCC pathway, a MatB pathway, and a mutase-differentiation Enzyme pathway.
The method for heterologously expressing epothilone according to claim 5 or 6, wherein the PCC pathway is supplemented by the addition of propionyl-CoA carboxylase; the MatB pathway is added by adding malonyl-CoA Supplemented with /methylmalonyl-CoA synthetase; the mutase-isomerase pathway is supplemented by the addition of methylmalonyl-CoA isomerase.
The method for heterologously expressing epothilone according to claim 7, wherein the propionyl-CoA carboxylase is Streptomyces coelicolor (S. Coelicolor) The accA1/pccB or pccA/pccB genes of A3(2).
The method for heterologously expressing epothilone according to claim 7, wherein the methylmalonyl-CoA isomerase is Streptomyces coelicolor (S. Coelicolor) The epi gene of A3(2).
The method for heterologously expressing epothilone according to claim 7, wherein the malonyl-CoA/methylmalonyl-CoA synthetase is Streptomyces coelicolor (S. Coelicolor) A3(2) matB gene.
The method of heterologously expressing epothilone according to claim 4, wherein a tRNA gene is further introduced into the host strain.
The method of heterologously expressing epothilone according to claim 11, wherein the tRNA gene is Arg anti-GCG, Arg One or more of the anti-TCG, Gln anti-CTG and Glu anti-CTC genes.
The method for heterologously expressing epothilone according to claim 12, wherein said Arg anti-GCG, Arg anti-TCG, Gln The anti-CTG and Glu anti-CTC genes are derived from Myxococcus xanthus DK 1622.
The method of heterologously expressing epothilone according to claim 12, wherein a promoter sequence is added in front of one or more genes in the epothilone gene cluster.
The method for heterologously expressing epothilone according to claim 14, wherein epoA, epoB, epoC, epoD, epoE and epoF in the epothilone gene cluster A promoter sequence is added before one or more of the 6 genes.
The method for heterologously expressing epothilone according to claim 14, wherein epoA, epoB, epoC, epoD, epoE and epoF in the epothilone gene cluster A promoter sequence was added before each of the 6 genes.
The method of heterologously expressing epothilone according to claim 15 or 16, wherein the promoter is PKan.
The method of heterologously expressing epothilone according to claim 17, wherein the promoter sequence is added by resplicing of the gene.
The method of heterologously expressing epothilone according to claim 18, wherein the splicing is carried out using a Bxb1 integrase splicing technique.
A genetically engineered strain heterologously expressing epothilone, characterized in that the genetically engineered strain incorporates an epothilone gene cluster and is supplemented with an epothilone precursor synthesis pathway.
The genetically engineered strain for heterologous expression of epothilone according to claim 20, wherein the basic strain of the genetically engineered strain is Burkholderiales DSM. 7029 strain.
The genetically engineered strain for heterologous expression of epothilone according to claim 21, wherein the basic strain is supplemented with a synthetic route of the epothilone precursor S-methylmalonyl-CoA.
The genetically engineered strain for heterologous expression of epothilone according to claim 22, wherein the S-methylmalonyl-CoA synthesis pathway comprises a PCC pathway, a MatB pathway, and a mutase-isomerization Enzyme pathway.
A genetically engineered strain for heterologous expression of epothilone according to claim 23, wherein in the base strain, Streptomyces coelicolor is added (S. Coelicolor) A3(2) accA1/pccB complements the PCC pathway; by adding Streptomyces coelicolor (S. Coelicolor) A3(2) epi gene complements the mutase-isomerase pathway; by adding Streptomyces coelicolor (S. Coelicolor) A3(2) matB gene complements the MatB pathway.
The genetically engineered strain for heterologous expression of epothilone according to claim 24, wherein a tRNA gene is further added to the base strain.
The genetically engineered strain for heterologous expression of epothilone according to claim 25, wherein the tRNA gene is Arg anti-GCG, Arg anti-TCG, Gln anti-CTG and Glu anti-CTC genes.
The genetically engineered strain for heterologous expression of epothilone according to claim 26, wherein a promoter sequence is added to one or more genes of the epothilone gene cluster.
The genetically engineered strain for heterologous expression of epothilone according to claim 27, wherein epoA, epoB, epoC, epoD, epoE and epoF in the epothilone gene cluster A promoter sequence was added before each of the six genes.
The genetically engineered strain for heterologous expression of epothilone according to claim 28, which is spliced in the order of epoA, epoB, epoC, epoD, epoE to epoF by resplicing to add the start Subsequence.
The genetically engineered strain for heterologous expression of epothilone according to claim 29, wherein the promoter is PKan.
A genetically engineered strain heterologously expressing epothilone, characterized in that the genetically engineered strain is Polyospora (Polyangium) Brachysporum)MMR11, deposit number CCTCC M 2017037, deposited on January 19, 2017 in China's typical culture collection management center.
A method for producing epothilone, characterized in that a strain according to any one of claims 19 to 30 is provided, which is fermented in a fermentation medium at 30 ± 2 °C.
A method for producing epothilone according to claim 32, wherein the fermentation medium is CYMG fermentation medium, and the formula per liter is casein 8 g, yeast extract 4 g, magnesium chloride hexahydrate 4.06 g, 50% glycerol 10 ml, trace elements 1 ml, sodium acetate 50 mg, sodium propionate 100 mg, methylmalonic acid 100 Mg, cysteine 2.5 mg, serine 5 mg, XAD-16 macroporous adsorption resin wet weight 1%, adjusted pH 7.0-7.5.
The method for producing epothilone according to claim 32, wherein the fermentation time is 3 days.